Development and validation of a liquid chromatography-tandem mass spectrometry assay for serum 25-hydroxyvitamin D2/D3 using a turbulent flow online extraction technology

Bunch, Dustin R.

Miller, Abby Y.

Wang, Sihe

Background: Vitamin D is important to health and disease. Liquid chromatography-tandem mass spectrometry (LC-MSMS) is considered the most accurate technology for quantification of serum 25-hydroxyvitamin D (25OHD) which is the best biomarker for estimating vitamin D nutritional status. Methods: Serum was mixed with acetonitrile containing hexadeuterated 25-hydroxyvitamin D3 (d6-25OHD3) and centrifuged 10 min at 15,634×g. The supernatant was injected onto a turbulent flow preparatory column then transferred to a polar endcapped C18 analytical column. The mass spectrometer was set for positive atmospheric pressure chemical ionization. Results: The analytical cycle time was 5.5 min. Inter- and intra-assay CV for both analytes across three concentrations ranged from 3.8% to 14.2%. The method was linear from 3.0 to 283.6 nmol/L for 25-hydroxyvitamin D3 (25OHD3) and 4.6 to 277.9 nmol/L for 25-hydroxyvitamin D2 (25OHD2), with an accuracy of 88.7%–118.7% and 90.7%–100.3%, respectively. No carryover or ion suppression was observed. Comparison with a radioimmunoassay using patient specimens (n=527) showed a mean difference of 5.2%, and diagnostic agreement of 80.9% with Deming regression of slope 0.867, intercept 12.8, standard error of estimate (SEE) 17.4, and r=0.8425. Conclusions: The LC-MSMS method coupled with turbulent flow technology for serum 25OHD quantitation is rapid, efficient, and suitable for clinical testing. Clin Chem Lab Med 2009;47:1565–72.